Chemokines are chemotactic cytokines that are released by a wide variety of cells to attract macrophages, lymphocytes, eosinophils, basophils and neutrophils to sites of inflammation (reviewed in Schall, Cytokine, 3:165-183 (1991), Schall, et al., Curr Opin. Immunol. 6:865-873 (1994) and Murphy, Rev. Immun., 12:593-633 (1994)). In addition to stimulating chemotaxis, other changes can be selectively induced by chemokines in responsive cells, including changes in cell shape, transient rises in the concentration of intracellular free calcium ions ([Ca2+]), granule exocytosis, integrin upregulation, formation of bioactive lipids (e.g., leukotrienes) and respiratory burst, which is associated with leukocyte activation. Thus, the chemokines are early triggers of the inflammatory response, causing inflammatory mediator release, chemotaxis and extravasation to sites of infection or inflammation.
There are two main classes of chemokines, CXC (alpha) and CC (beta), depending on whether the first two cysteines are separated by a single amino acid (C-X-C) or are adjacent (C-C). The alpha-chemokines, such as CXCL1 (GROα) and CXCL8 (interleukin-8, IL-8) are chemotactic primarily for neutrophils, whereas beta-chemokines, such as CCL5 (RANTES) and CCL20 (LARC, MIP-3α), are chemotactic for T cells, B cells, macrophages, eosinophils and basophils (Deng, et al., Nature, 381:661-666 (1996)). The chemokines bind specific cell-surface receptors belonging to the family of G-protein-coupled seven-transmembrane-domain proteins (reviewed in Horuk, Trends Pharm. Sci., 15:159-165 (1994)) which are termed “chemokine receptors.”
On binding their cognate ligands, chemokine receptors transduce an intracellular signal through the associated trimeric G protein, resulting in a rapid increase in intracellular calcium concentration. There are at least eleven human chemokine receptors that bind or respond to beta-chemokines and at least seven human chemokine receptors that bind to the alpha chemokines. Additionally CX3CR1 (fractalkine receptor) can bind to the fractalkine chemokine, which is distinguished by a series of three amino acids between the first two cysteines. Chemokine receptors, have been implicated as being important mediators of inflammatory and immunoregulatory disorders and diseases, including asthma and allergic diseases, as well as autoimmune pathologies such as rheumatoid arthritis and atherosclerosis.
The chemokine receptor CCR6 is known to be expressed by memory (but not naïve) CD4 T cells, IL17-secreting αβ T cells, IL17-secreting γδ T cells, regulatory T cells, B cells and dendritic cells. Its only known ligand is CCL20 (MIP-3α, LARC), for which it shows strong binding. It is expressed on approximately 30-60% of adult peripheral blood effector/memory CD4+ T cells. CCR6 is involved in leukocyte homing to inflamed tissue, particularly the skin, lungs and gut; and is co-expressed on a subset of T cells that have a skin homing phenotype (i.e., T cells that express the cutaneous lymphocyte antigen (CLA) and CCR4). Thus CCR6 may be an important player in skin pathologies in which leukocytes participate.
CCR6 expression has been linked to psoriasis. In humans, a large majority of IL17-expressing skin-homing CD4 T cells in the peripheral blood express CCR6 (Homey, et. al., JI, 2000). IL17 secreting cells are central agents in several inflammatory diseases. T cells, such as γδ T cells and TH17 T cells produce IL17 after activation. The pathogenic effects of IL17 have been associated with human diseases such as rheumatoid arthritis (Patel D D et. al., Ann Rheum Dis 2013), multiple sclerosis (Zepp J, Wu L, and X Li Trends Immunol 2011), and psoriasis (Martin D A et. al., J Invest Dermatol 2012). Evidence strongly linking IL17 with psoriasis include gene wide association studies that show strong association between psoriasis and genes upstream (IL-23) or downstream (NFκb) of IL17 signaling pathways as well as efficacy in targeting IL17 in a clinical setting (Martin D A et. al., J. Invest Dermat. 2012; Papp et. al., NEJM, 2012; Papp et. al., NEJM, 2012). In addition to enhanced CCL20-mediated chemotaxis, CCR6+ T cells isolated from psoriatic patients preferentially secrete IL-17A, IL22, and TNFα when compared to healthy controls (Kagami, et. al., J. Invest. Dermatol., 2010). Lastly, ccl20 mRNA was up-regulated in lesional psoriatic skin samples (Homey, et. al., JI, 2000; Dieu-Nosjean, et. al., JEM, 2000). In mice, CCR6 knock-out mice were protected from IL-23 driven psoriasis (Hedrick M. N. et. al. JCI, 2009). Thus, a multitude of evidence in both mice and men suggest a protective role for CCR6 blockade in psoriasis and psoriasis-like models.
CCR6 is also expressed by dendritic cells at critical stages during their development, and is important for their migration through tissues (Sozzani et al, J Leuk Biol, 66:1, 1999). Dendritic cells are responsible for presenting antigens to T cells within lymph nodes, and thus inhibition of dendritic cell trafficking can have a dampening effect on T cell mediated inflammatory responses (Banchereau and Steinman, Nature, 392:245, 1998).
CCR6 is expressed by B cells, and it has recently been demonstrated that CCR6-mediated B cell migration is required for B cells to engage is memory responses to soluble antigen (Elgueta et al., J Immunol, 194:505, 2015). Inhibiting such B cell migration via CCR6 blockade can therefore potentially dampen B cell mediated (and therefore antibody-mediated) inflammatory responses in disorders such as lupus, rheumatoid arthritis and pemphagus.
CCR6 is often expressed by colorectal cancer (CRC) cells. High expression of this receptor is associated with poor outcome for CRC patients, and CCR6 itself has been proposed to contribute to migration of CRC cells leading to metastasis (Liu J. et. al. PLOSone 20149 (6):e101137).
Chemokines that bind to a separate receptor, CXCR2, promote the accumulation and activation of neutrophils. These chemokines are implicated in a wide range of acute and chronic inflammatory disorders such as psoriasis, rheumatoid arthritis, radiation-induced fibrotic lung disease, autoimmune bullous dermatoses (AIBD), chronic obstructive pulmonary disease (COPD) and ozone-induced airway inflammation (see, Baggiolini et al., FEBS Lett. 307:97 (1992); Miller et al., Crit. Rev. Immunol. 12:17 (1992); Oppenheim et al., Annu. Rev. Immunol. 9: 617 (1991); Seitz et al., J Clin. Invest. 87: 463 (1991); Miller et al., Ann. Rev. Respir. Dis. 146:427 (1992); and Donnely et al., Lancet 341: 643 (1993), Fox & Haston, Radiation Oncology, 85:215 (2013), Hirose et al., J Genet. Syndr. Genet. Ther. S3:005 (2013), Miller et al., Eur. J Drug Metab. Pharmacokinet. 39:173 (2014), Lazaar et al., Br. J Clin. Pharmacol., 72:282 (2011)).
In addition to inflammatory disorders, some of the CXCR2 ligand chemokines including CXCL1, CXCL2, CXCL3, and CXCL5 have been implicated in the induction of tumor angiogenesis (Strieter et al. JBC 270: 27348-27357 (1995)). Some CXCR2 ligand chemokines are exacerbating agents during ischemic stroke (Connell et al., Neurosci. Lett., 15:30111 (2015). Their angiogenic activity is possibly due to the activation of CXCR2 expressed on the surface of vascular endothelial cells (ECs) in surrounding vessels by the chemokines.
Many types of tumors are known to produce CXCR2 ligand chemokines. The production of these chemokines correlates with a more aggressive phenotype (Inoue et al. Clin Cancer Res 6:2104-2119 (2000)) and poor prognosis (Yoneda et. al. J Nat Cancer Inst 90:447-454 (1998)). As the chemokines are potent chemotactic factors for EC chemotaxis, they probably induce chemotaxis of endothelial cells toward their site of production in the tumor. This may be a critical step in the induction of tumor angiogenesis. Inhibitors of CXCR2 will inhibit the angiogenic activity of the ELR-CXC chemokines and therefore block the tumor growth. This anti-tumor activity has been demonstrated for antibodies to CXCL8 (Arenberg et al. J Clin Invest 97:2792-2802 (1996)), ENA-78 (Arenberg et al. J Clin Invest 102:465-72 (1998)), and CXCL1 (Haghnegahdar et al. J. Leukoc Biology 67:53-62 (2000)).
Many tumor cells express CXCR2 and tumor cells may stimulate their own growth by secreting ELR-CXC chemokines. Thus, in addition to decreasing angiogenesis within tumors, CXCR2 inhibitors may directly inhibit the growth of tumor cells.
CXCR2 is often expressed by myeloid-derived suppressor cells (MDSC) within the microenvironment of tumors. MDSC are implicated in the suppression of tumor immune responses, and migration of MDSC in response to CXCR2 ligand chemokines is most likely responsible for attracting these cells into tumors (Marvel and Gabrilovich, J. Clin. Invest. 13:1 (2015) and Mackall et al., Sci. Trans. Med. 6:237 (2014)). Thus, CXCR2 inhibitors may reverse suppressive processes and thereby allow immune cells to more effectively reject the tumor. In fact, blocking the activation of CXC-chemokine receptors has proven useful as a combination therapy with checkpoint inhibitors in suppressing tumor growth, suggesting that CXCR2 blockade may also enhance tumor rejection in combination with other anti-tumor therapies, including but not limited to vaccines or traditional cytotoxic chemotherapies (see Highfill et al., Science Translational Medicine, 6:237 (2014)).
The activities of CCR6 and CXCR2 have each been associated with poor outcome in certain cancer types including CRC, although each is likely to work through a different, potentially complementary, mechanism (Nandi et al., PLoS One, 9:e97566, 2014; Liu et al, PLoS One, 9:e101137, 2014; Cheluvappa, Int J Colorectal Dis, 29:1181, 2014; Zhang, Biomed Pharmacother. 69:242, 2014; Lee et al, Int J Cancer, 135:232, 2014; Wang and DuBois, Oncoimmunology, 29:e28581, 2014; Wu et al, Int J Clin Exp Med, 8:5883, 2015).
In view of the clinical importance of CCR6 and CXCR2, the identification of compounds that modulate the function of one or both of these two receptors represent an attractive avenue into the development of new therapeutic agents. Such compounds and methods for their use are provided herein.